1. Field of the Invention
The invention is a method for producing and/or using arrays which contain a multiplicity of compounds for the purpose of screening to identify molecules with desirable properties. The various compounds forming the elements of the array can be synthesized in place using combinatorial synthesis schemes, or pre-synthesized compounds can be incorporated into the array. Applications to genetic screening, in vitro diagnostics, and drug discovery are anticipated.
2. Discussion of the Prior Art.
Current trends in medical diagnostic testing and pharmaceutical research are toward conducting a large number of tests concurrently on a single device. For example, one such device has been called a DNA xe2x80x9cchipxe2x80x9d for sequence analysis. A DNA chip contains a large number (thousands) of unique DNA molecules (probes) immobilized on a flat surface in the form of an array (e.g. checker board). The company Affymax uses a photo-lithographic method to produce DNA chips (Fodor, S.P.A., et.al., Science, 1991, 251, 767-773). Another approach is to use pre-synthesized molecules which are applied and immobilized to a suitable substrate (e.g. microporous membrane).
For example, an unknown sample of DNA (target) is applied to the chip and a hybridization pattern is formed. The pattern is indicative of the strength of interaction between the target DNA and the various immobilized probes and can yield sequence information. When the sequence of the target DNA is not known the technology is generally referred to as sequencing by hybridization (SBH) as described in U.S. Pat. No. 5,202,231. In other applications where the sequence of the target is known and detection is directed at identification of a change associated with a disease state the method is commonly referred to as xe2x80x9cre-sequencingxe2x80x9d or allele specific oligonucleotide hybridization.
Another trend in the arena of pharmaceutical drug discovery is known as combinatorial synthesis. In this case a large number of similar compounds are synthesized and simultaneously screened for the desired biological response, for example binding to a receptor molecule. When one or more candidate compounds in the combinatorial bank are discovered they are synthesized in larger quantities for further testing. Molecules of interest include peptides, nucleic acids as well as drug compounds synthesized by standard organic chemical methods or other novel methods for drug discovery.
Finally, in the area of in vitro diagnostics there is a need for panel assays where several tests are run concurrently on a given sample using an array of immobilized binding agents. An example of such array immunoassay devices is described in U.S. Pat. Nos. 4,591,570 and 4,829,010. Pre-manufactured compounds, such as mono-clonal antibodies, are used to make arrays whose elements have particular binding properties for diagnostic analysis. In principle a patient test sample can be simultaneously analyzed for the presence or absence of several molecules, i.e. analytes. Further, the levels of the various analytes can be measured simultaneously by quantitative analysis of the signals developed at each site of the array. In other applications there is a need for graphic symbols that can be visually analyzed to determine the presence or absence of a single analyte in a patient test sample. For the present invention, the graphic symbol can be thought of as an array of individual elements that are spatially arranged to yield a graphic symbol as a result of the detection process. In this case, the size of the array elements determine the xe2x80x9cgrainxe2x80x9d of the graphic symbol.
Thus, there is a need for methods to produce and concurrently test multiple compounds or binding agents in the form of an array. An additional requirement is the need for high density devices (i.e. high spatial density) so that the large numbers of compounds are presented in a package of reasonable size. For example, a device that contains a possible 8-mer DNA sequences composed of the 4 DNA bases, A (adenine), T (thymine), G (guanine) and C (cytosine) requires 48=65536 different compounds. If each element (i.e. a zone of immobilized binding agent) was a square only 1 millimeter (1 mm=0.1 cm) in size, an array of 65536 elements would be 10 inches on a side. Clearly, such devices would be difficult to manipulate and would require relatively large amounts of the test sample to be spread evenly over the array surface. A 0.1 mm thick layer of test sample spread on a 10xc3x9710 inch area amounts to about 6.5 ml (ml=milliliter=6.5xc3x9710-3 liter). Since most test samples are of biological origin, they are typically very expensive, difficult to prepare and in short supply. Examples of test samples are PCR products or purified drug receptors which are typically available in microliter quantities-1000 times less than in the above example. In most cases, DNA synthesis requires the use of expensive components, phosphoramidite DNA synthesis being a case in point, so surface area of the array is also important during the manufacturing step.
Thus, the smaller the size of the array elements involved in the synthesis the more economical the device will be to produce and use. Several methods exist to create chips with large numbers of different sequences but often result in devices with large features, large physical size and, hence, low spatial density. For example, one method uses disks or channels to produce arrays of probe DNA""s using standard DNA synthesis chemistry (see for example, Williams, et.al., Nucleic Acids Research 1994, 22, 1365 or Southern et.al., Nucleic Acids Research, 1994, 22, 1368 and references therein). The drawback of this method is that small feature size is not obtained.
Another method of making DNA chips is to use pre-synthesized probe DNA""s and a printing device to allow application of the various compounds. The probes are applied to the chip with a pin or a pipette in the pattern of an array and immobilized by any of a variety of techniques such as adsorption or covalent linkage. An example of such DNA arrays is described in Stimpson et.al. Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6379-6383, July 1996. Since elements of the array are formed by the application of a DNA solution to the surface of the array the process is relatively slow.
One method to produce high density chips uses photo-lithography (Pease et al., Proc. Natl. Acad. Sci. USA Vol. 91, pp. 5022, 1994). One drawback to this method is that it relies on a new DNA synthesis chemistry as opposed to the standard phosphoramidite chemistry used in commercial DNA synthesizers. The technology feeds off the methods evolved in the electronics industry and therefore has some of the same requirements, vis, accurate positioning to micron scales, clean room requirements and the use of multiple photo-masks to define the array pattern. Although electronic xe2x80x9cchipsxe2x80x9d (for example an Intel PENTIUM(copyright) microprocessor) are mass produced economically, they are typically too expensive to be used as a disposable element, as is needed with a DNA chip.
Another drawback of such chips is the use of a solid impermeable support material like a glass slide or cover slip. As a result, only the very small amount of material immobilized on the surface of the solid chip is used to capture target molecules. An improvement is described using porous silicon or channel glass whereby hybridization reactions occur within the three-dimensional volumes of porous silicon dioxide of channel array glass rather than two-dimensional surface areas (Beattie, K. L., The 1994 San Diego Conference: The Genetic Revolution).
Unfortunately, all the array fabrication methods mentioned above suffer from a common limitation, i.e., each element of each array is a unique synthesis or an application step. This is true even when array elements or entire arrays are simply duplicated or produced xe2x80x9cin parallelxe2x80x9d, or more accurately, concurrently. Since each element is a unique synthesis or application there is a chance for variation between corresponding elements on different arrays or, for that matter, duplicated elements on the same array. When multiple arrays are produced concurrently it is carried out in a two dimensional fashion, i.e., arrays are produced next to one another in a two dimensional X-Y plane, where X and Y refer to the two degrees of spatial freedom. In a photo-lithographic process, increasing the number of chips on a wafer (the substrate on which multiple arrays are produced) results in an increase in surface area which increases demand on the chemicals used in photo-chemistry (assuming no change in chip size).
The object of the present invention is to extend array construction into a third dimension, Z, so that each array element formed by a synthesis or application of a binding agent is used to produce many arrays. Individual arrays are formed by cutting slabs along the Z axis of a bundle assembled from the various array elements.
The present invention forms elements for the construction of two dimensional (X-Y) arrays by synthesis or applications of binding agents in a third or Z dimension. The invention is based on the observation that arrays cut from bundles of porous rods or spiral wound porous sheets behave like membranes composed of said porous materials and conduct flow through the multitude of edges exposed during cutting. Surprisingly, liquid flows substantially through the multiple porous rod or sheets which comprise the array and not through the intervening spaces between the array elements.
In one embodiment, the elements of the array are formed by the ends of rods of porous materials which are compatible with a chemical synthesis or compound application step. For application of protoinaccous (e.g. antibodies) or nucleic acid (e.g. derived from a cDNA library) compounds the porous matrix can be selected from any of the materials currently used to produce microporous membranes by a phase inversion or a leaching process. Examples of suitable microporous membrane materials are cellulose, nitrocellulose, polysulfone, nylon, polypropylene or glass. (For a discussion on methods of producing microporous polymer membranes see Synthetic Polymeric Membranes, R. E. Kesting, John Wiley and Sons, 1985, ISBN 0471-807176. For a discussion on methods of producing porous glass materials see Solid Phase Biochemistry, W. E. Scouten Ed., John Wiley and Sones, 1983, ISBN 0471-08585-5, Chapter 11, Application of Controlled Pore Glass in Solid Phase Biochemistry by W. Haller). In particular, nitrocellulose is preferred for protein components while nylon is preferred for immobilization of nucleic acid compounds. In each case, rods or xe2x80x9cthreadsxe2x80x9d of such materials can be formed from processes similar to those used in producing hollow fiber membranes or flat sheet membranes. Alternatively, materials that are commonly used for producing xe2x80x9cthreadsxe2x80x9d or xe2x80x9cyarnsxe2x80x9d by a spinning process can be utilized to make rods, for example, polyester thread. Each rod is dipped or otherwise exposed to a unique binding agent to allow uniform attachment throughout its length (Z axis). The attachment procedure may involve simple adsorption, covalent attachment chemistries, multiple washing or adsorption steps or other manipulations to achieve the desired properties of the particular array element. For a complete discussion on methods of covalent attachment to various solid supports see Pierce Catalog and Handbook, 1994. Synthesis steps on the rods elements may involve standard DNA synthesis chemistries or other synthetic methods of organic chemistry to achieve the desired spectrum of molecules for screening as used in drug discovery or gene identification. It is important to note that the binding agent is introduced to the array element in a batch mode, i.e., the entire rod is treated uniformly. The array elements can be subjected to a quality control step before assembly into the bundle used to make the arrays. When all array elements are available they ere formed into a rod bundle using radial compression about the Z axis of the bundle. The rods may be organized in the bundle by using a guide, i.e., a plate with a series of holes to direct the rods to a particular point of the array. The bundle can be compressed by pulling it through a cone shaped guide. A sheath is wrapped around the bundle, as in the insulation around a bundle of conducting electrical wires, to hold the elements in place. The resulting rod bundle is then sliced into multiple arrays along the Z axis. Each array consists of a two dimensional arrangement of rod elements with the various compounds displayed on the newly cut ends of each rod. Hence, each synthesis or application step to produce a given array element is used to produce many arrays. Uniformity of binding agent is anticipated for a given batch of arrays because any given element is produced by a single synthesis or application step. This is opposed to prior art where each element is produce by a unique application or synthesis, even when said reactions are carried out in parallel. Another interesting feature of the method is that array density is determined by the diameter of the rod elements and is not limited by the density of the reagent application or synthesis (e.g. ink jet or photo-lithography).
In some cases it may be desirable to arrange particular rod elements in the form a graphic symbol within the array. To form a graphic symbol from rod elements a guide device would be used to direct each rod element to the correct position in the array to form the graphic symbol. For a graphic symbol large enough to be read by the human eye, multiple rods with the same binding properties are used. The diameter of the rods at the edge of the symbol determines the grain or pixel density of the final graphic. For a production process, each rod element is stockpiled on a spool. The spools are dipped or otherwise reacted to introduce the desired immobilized binding agents. The spools are fed into the guide and pulled through to form a rod bundle with the appropriate spatial arrangement of rod elements. A sheath is applied to the rod bundle as it emerges from the guide and the bundle is either wrapped on a new larger spool or cut into convenient lengths for storage or directly cut into slab arrays. To create a xe2x80x9cplusxe2x80x9d symbol, a guide with the xe2x80x9cplusxe2x80x9d configuration in the center (i.e. a cruciform hole) with 4 surrounding areas in each quadrant formed by the xe2x80x9cplusxe2x80x9d is used. Multiple Rods from spools treated with the appropriate binding agents for a particular assay are fed into the xe2x80x9cplusxe2x80x9d portion of the guide while multiple non-binding rods are fed into the adjacent guides formed by the 4 quadrants. Of course, preformed bundles of rods (i.e. yarns) could be used to feed the quadrant portions of the array. The process is similar to that described by American Filtrona (Richmond, Va.) and has been given the name xe2x80x9cpulltrusionxe2x80x9d. In this fashion, arrays with binding agents or ink printing distributed in various graphic arrangements can be manufactured.
In another embodiment, the elements of the array are formed by the edges of porous sheet materials, such as microporous membranes. The binding agents are applied or synthesized on the sheet to create binding zones of a given binding agent along the entire edge of the sheet. Alternatively, thin lines of compounds are applied or synthesized to create multiple array elements on a single sheet of material. In this case, each line of immobilized binding agent printed on the sheet is akin to a rod in the aforementioned example. Printing a line of binding agent on the porous sheet results in migration of the agent into the matrix of the material. When the sheet is cut, the impregnated agent in the matrix of the sheet is exposed on the freshly cut edge. The manufactured dimension of the array element is given by the thickness of the membrane material and the width of the line used to apply the compounds. However, depending on the distribution of binding agent within the sheet material the actual dimension of the array element may be smaller, for example, if the binding agent only penetrates xc2xd the thickness of the sheet. In some special circumstances it may be possible to apply or synthesize binding reagents on both sides of a sheet to further to farther increase array density. After the desired compounds are present on the sheets, the sheets are assembled into stacks or rolls before cutting into individual arrays. Individual arrays are generated by cutting slabs as thin as possible along the Z axis of the roll or the stack. As in the rod format, each array element is incorporated into multiple arrays. Since one dimension of the array element is determined by the sheet thickness, reasonably high density arrays can be obtained even with low density reagent application methods.
An advantage of the printed line format over the rod element format is that the spatial arrangement of the array elements (i.e. printed lines) is fixed along the membrane sheet whereas the rods must be guided to known positions. An advantage of the rod method over the sheet format is that the manufacturing process for the former is compatible with a continuous process while the latter is more suitable to a unit process.
In some cases it may be desirable to use an adhesive compound to bind either the sheets in a stack or the layers of a rolled sheet together to from a cohesive structure. The adhesive used for this purpose must not migrate during the cutting process used to form the individual arrays or else the edges of the sheet material become covered with adhesive and are not accessible to test solutions. Suitable adhesives for binding the sheets are heat activated-double sided DOW Adhesive Films (Dow Chemical, Midland, Mich.). The important features of adhesive selection are: (1) the adhesive does not wet and thereby occlude the pores of the sheet material before and during setting (2) the adhesive sets to a substantially solid consistency that does not migrate and cover the sheet edges during cutting (3) the set adhesive is not brittle and susceptible to cracking when the individual arrays are released from the bundle or roll and (4) the adhesive is stable to the aqueous solvent of the test sample. In general, pressure sensitive adhesives (e.g. SCOTCH TAPE(copyright) adhesive, 3M, St. Paul, Minn.) are not desirable because of adhesive migration during mechanical cutting. However, other cutting methods using lasers may allow the use of pressure applied adhesives. One advantage of the roll format over the stack format is that, typically, the compressional forces supplied by the sheath in the rolled structure are sufficient to maintain the integrity of the individual arrays cut from the roll without using any adhesive. This is true for both rod bundles and spiral sheet bundles.
The array is then used to carry out screening tests or diagnostic evaluations. In one procedure, a test sample is exposed to all the array elements by application of a small volume of liquid to the top of the array surface. The liquid is drawn into the array by capillary action. To improve sensitivity, more fluid can be drawn through the array by setting it on an absorbent pad. A washing step can be incorporated to remove unbound components of the test sample from the array. Array elements which have affinity for components of the test sample will retain said components by specific binding events. In a sense, the array acts as a membrane which is composed of short segments of the porous materials used to make the elements of the array. In another procedure, the array is simply soaked in a test sample and the binding reaction proceeds by diffusion of target molecules to the surface of the array or into the matrix of the array. A sample contains the component or components whose binding patterns are to be identified by use of the array. For example, samples may be derived from PCR amplification of materials collected from a human patient, plant or other organism, the products of a combinatorial synthesis, a prospective drug, an antibody or mixture of antibodies, or a volume of blood, plasma, serum or urine. In each case, those skilled in the art recognize the need for the appropriate sample preparation before applying the sample to an array for binding analysis. For example, filtration, centrifugation, or addition of agents to prevent non-specific binding are among the possible operations in preparation of the sample before it is introduced to a diagnostic test.